Electrically mediated gene transfer, also termed DNA electrotransfer or electrogenetherapy, uses various single or multiple-electrode designs such as arrays of two or more electrodes that typically are designed as needle electrodes for insertion into a tissue, said electrode being connected to a pulse generator. The method has been shown to be effective to electrotransfer plasmid DNA to various tissues: muscles, liver, skin, tumors, mouse testis, etc. . . .
Mechanisms by which electric pulses mediate DNA transfer into target cells are not well understood. Nevertheless, there is a common agreement that for an improved DNA transfer into tissues, cells in that tissue must be permeabilized. For the years 1960-1970, in vitro studies showed that pulsed electric fields (PEF) delivery on living cells induce a reversible or irreversible breakdown of the cell membranes, called electropermeabilization. Such a permeabilization can be achieved using simple runs of short square wave electric pulses (in the range of 100 [mu]s). This kind of pulses has been widely used for the local delivery of non-permeant anticancer drugs (like bleomycin or cisplatin) in a treatment termed 'antitumor electrochemotherapy. Indeed, the delivery to tumors of e.g. 8 pulses of 1300 V/cm and 100 [mu]s either in vitro or in vivo is sufficient to induce transient rearrangements of the cell membrane that allow non-permeant anticancer molecules like bleomycin to enter the cell by diffusion and to fully exert their cytotoxic activity.
These short permeabilizing electric pulses have also been shown to increase the transfer of plasmid DNA into several tissues. However, another type of square-wave electric pulses was applied to muscles, tumors, liver and some other tissues, and was found to be more effective for DNA electrotransfer. These pulses usually are of lower voltage but much longer duration (in the range of tens of milliseconds). It is assumed that this type of pulses or combination of pulses (in the range of 100 μs to 100 ms and 25 to 1500 V/cm) mediate DNA transfer into the cells by inducing two distinct effects that include cell permeabilization (like the short pulses) and DNA electrophoretic migration during the delivery of the electric field. This technique, called gene electrotransfer is used to internalize DNA plasmids in cells without causing irreversible damages on plasma membranes. Efficient electrotransfer into cells has been described in WO-A-99/01158 and in WO-A-98/43702 notably.
A new kind of PEF, nanosecond pulsed electric fields (nsPEF) is actually under study. nsPEF are ultra-short pulses (10 ns, or even less than 10 ns to 300 ns) with higher electric field strength (10 to 150 kV/cm or more) that do not increase the temperature of the exposed cells. First studies showed that nsPEF induced permeabilization of intracellular membranes (granules, vesicles, mitochondria, nucleus . . . ) but not of plasma membrane.
nsPEF also have been shown to induce a release of intracellular calcium from the endoplasmic reticulum in cells under conditions maintaining plasma membrane integrity. Differential effects in cells exposed to ultra-short, high intensity electric fields have been studied by means of cell survival, DNA damage, and cell cycle analysis. nsPEF also have been shown to induce an enhancement of gene transfection efficiency. Within these studies, one experiment showed that the application of 1 nsPEF (10 ns, 150 kV/cm) 30 min after the GFP gene electrotransfer into cells in suspension allows an increase of 3-fold of the GFP expression compared to electrotransfer only. As the electrogenetransfer, like the other approaches for non viral gene therapy, is considered less efficient than the viral approaches for gene therapy, an increase of 3-fold or more of the GFP reporter gene expression is very important for the development of this non-viral gene therapy approach, which is considered, in general, safer and easier than the viral approaches.
Moreover, electroporation has been applied for delivering molecules to subsurface tissues using various single or multiple-electrode designs such as arrays of two or more electrodes that typically are designed as needle electrodes for insertion into said tissue, said electrode being connected to a pulse generator. Generally, such arrays define a treatment zone lying between the needle electrodes of the array. Such treatment zones therefore comprise a three dimensional volume of tissue wherein cells within the treatment zone are exposed to an electric field of an intensity sufficient to cause temporary or reversible poration, or even sometimes irreversible poration, of the cell membranes to those cells lying within and or near the three dimensional volume. The U.S. Pat. No. 5,674,267 discloses such a process and an electric pulse applicator for the treatment of biological tissue applying an electric field to the cells of biological tissue to modify the properties of their membranes.
Current practices for electroporating cells in tissue include use of significant voltages in order to impart through the three dimensional treatment zone a relatively uniform electric field. By “relatively uniform” is meant that electric lines of force coincident with application of an electric pulse sufficient to cause poration is imparted across the cells somewhat evenly throughout the three dimensional treatment zone volume. Besides the invasive aspect of a device with multiple needles, typical electroporation techniques, as stated above, result in variability in electroporation of cells within a treatment zone. This is a drawback to medical use of electroporation in that dispersion of treatment molecules of the injected bolus into surrounding tissue results in loss of control as to the amount of such treatment molecule that is ultimately transfected into cells within the treatment zone by the electroporation event.
Moreover, the use of metallic electrodes on contact of the skin or of the biological tissues may cause burns which are visible on the skin and which can be painful for a patient. These burns are probably of electrochemical kind. Indeed, the oxidizable metal of electrodes and the molecule of H2O and NaCl present in the surrounding of electrodes and on contact of said electrodes create various reactive species when the pulses are delivered. To avoid, or to reduce these burns, it is necessary to use biocompatible materials, for example specific metals or alloys, to elaborate the electrodes. This constraint may preclude the use of materials with optimal electrical properties (conductivity, permittivity) that may contain heavy metals, toxic ions, or, in general, non biocompatible substances. The electrochemical burns may affect normal cells reducing the efficacy of the electrogenetransfer or reducing the volumes treated by electrochemotherapy (as the electric pulses by themselves does not kill the cells in this application, and the bleomycin is killing almost exclusively the malignant tumor cells and sparing the non-dividing normal cells). Moreover, the ultrashort nanopulses seem to be unable to provoke the contraction of the muscles located in the contact or close to the electrodes, which can add comfort to the patient with respect to the treatment by electrochemotherapy using classical 100 μs-long pulses.
To overcome these drawbacks, it has been already imagined using insulated electrodes to deliver electric pulses onto any organic or inorganic conductive material and/or any biological material and/or to cells in vivo, ex vivo or in vitro, for example for the electroporation of the cells, for the electrically mediated transfer gene transfer of nucleic acids into tissue cell using a pulsed electric field and/or for the electromanipulation, in general, of the cell membrane or of the cell inside. Such insulated electrodes are disclosed in the European patent application EP08290714.8 filed Jul. 21, 2008 by the applicant. Said electrode includes a conductive main body and an electrically insulating coating and is intended to be introduced into and/or at the vicinity of a conductive material to be treated, for an electric pulse applicator for the treatment of conductive material, said electric pulse applicator comprising a pulse generator sending pulses to the electrodes having a slope (dE/dt) greater than 1015 V/m/s. In these conditions of pulse, the electrically insulating coating of electrodes looses its insulating properties allowing the generation of a “nanopulsed” electrical field.
The electrodes are usually rigid and machined with a cutting tool or molded before coating in such a manner that the shapes and the dimensional accuracy are limited, more particularly for electrodes of small dimensions. Consequently, there is a need in one hand for a method for producing rigid or flexible insulated electrodes with any desired shape, including 3D shape, and dimensions, including small dimensions, and on the other hand for a device allowing the real time observation of the effect of high electric field on material biological.